Graft copolymer for cation- exchange chromatography

Abstract

The invention relates to chromatographic separating materials having improved binding capacity for biological constituents in cell culture supernatants, or animal or human body fluids, in particular for monoclonal antibodies. The present invention likewise relates to the preparation of separating materials of this type, and to the use thereof, in particular for the removal of charged biopolymers from corresponding liquids.

Description

[0001]The invention relates to a separating material having improved binding capacity, to the preparation thereof, and to the use thereof for the removal of charged biopolymers from liquids.PRIOR ART[0002]Chromatography is one of the most suitable methods for the isolation of proteins. Monoclonal antibodies can be purified, for example, by affinity chromatography using protein A ligands. Binding to the ligand from the cell culture supernatant is possible without adaptation of pH and salt concentration. Nevertheless, these sorbents can only be employed to a limited extent due to their high costs and due to bleeding-out of the ligand.[0003]The use of high-capacity ion exchanger resins is a favourable alternative. However, the conduction value in the cell culture supernatant must be reduced in order that binding to the ion exchanger takes place. This can be carried out by desalination or by dilution of the supernatant. Both possibilities are undesired, in particular, in the case of lar...

AI Technical Summary

Benefits of technology

[0220]The separating material according to the invention can be used in a first chromatographic purification step (capture step) of a work-up process for a biopolymer. It is normally advantageous for the solid-containing crude solutions, such as, for example, cell suspensions or cell homogenates, firstly to be filtered before the capture step in order to remove coarse impurities, such as entire cells or cell debris. An advantage of the present invention, as described above, consists in that the ion strength of the cell culture supernatant does not have to be adapted. The capture step is generally followed, if the desired purity of the biopolymer has not yet been achieved, by further chromatographic purification steps using other separating materials which are capable of removing the various residual impurities. Since the sequence in which the separating materials are used may have an influence on the overall performance of the process, it may in certain cases be advantageous not to employ the separating material according to the invention until the second, third or fourth purification step.

[0221]The invention likewise relates to a kit for the purification or separation of biopolymers from one or more other substances in a liquid. The kit consists of a chromatography column which is packed with the separating material according to the invention, one or more buffers and a pack leaflet with written instructions. The liquid is adjusted to a pH of, for example, 5.5 using a buffer and brought into contact with the chromatography column. The column is firstly washed with a wash buffer, giving one fraction of the non-binding constituents, and the biopolymers are then desorbed using an elution buffer of higher ion strength, for example using 1 M NaCl solution, and obtained in a second fraction.

[0222]The present description enables the person skilled in the art to apply the invention comprehensively. Even without further comments, it is therefore assumed that a person skilled in the art will be able to utilise the above description in the broadest scope.

[0223]If anything is unclear, it goes without saying that the publications and patent literature cited should be consulted. Accordingly, these documents are regarded as part of the disclosure content of the present description.

[0224]For better understanding and in order to illustrate the invention, examples are given below which are within the scope of protection of the present invention. These examples also serve to illustrate possible variants. Owing to the general validity of the inventive principle described, however, the examples are not suitable for reducing the scope of protection of the present application to these alone.

[0225]Furthermore, it goes without saying to the person skilled in the art that, both in the examples given and also in the remainder of the description, the component amounts present in the compositions always only add up to 100% by weight or mol %, based on the composition as a whole, and cannot exceed this, even if higher values could arise from the percent ranges indicated. Unless indicated otherwise, % data are % by weight or mol %, with the exception of ratios, which are shown in volume data, such as, for example, eluents, for the preparation of which solvents in certain volume ratios are used in a mixture.

Problems solved by technology

Nevertheless, these sorbents can only be employed to a limited extent due to their high costs and due to bleeding-out of the ligand.

Both possibilities are undesired, in particular, in the case of large-volume production processes.

However, there are only a few commercially available adsorbents which are suitable for binding proteins and in particular antibodies from cell culture supernatants.

However, this gel was not developed especially for antibodies and binds 45 mg / ml of BSA.

In addition, there are further commercially available products containing synthetic ligands which are only able to bind antibodies from buffer solutions.

On use for the treatment of cell culture supernatants, unsatisfactory or no binding are obtained.

This result is probably caused by components in the cell culture supernatant which have an interfering behaviour.

However, no separating materials are known to date which can be prepared by a process which is simple to carry out using inexpensive starting materials and which have such high separation activities, in particular with respect to monoclonal antibodies, on use for the separation of cell culture supernatants or other biological liquids without significant reduction in the conductivities that they would be suitable for use on an industrial scale.

Consequently, too little protein can be bound to the derivatised separating material obtained.

Although the second preparation variant has, as a two-step process, the disadvantage of an additional reaction step, the graft polymerisation is, however, not restricted by the efficacy of the added cosolvent.

However, a disadvantage of this procedure in the case of reactions on an industrial scale is a considerable increase in the costs of the two-step preparation.

For economic reasons, this procedure is therefore not a true alternative.

However, the carboxyl groups generally cannot always be reacted completely here at high graft polymer densities.

Owing to the general validity of the inventive principle described, however, the examples are not suitable for reducing the scope of protection of the present application to these alone.

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